The value of achieving a quantitative, mechanistic understanding of how proteins perform their functions is well appreciated for protein classes such as enzymes, ion channels and G protein-coupled receptors, and good tools and approaches for such work are well established for these systems. In contrast, our quantitative understanding of the function of another medicinally important class of proteins - that is the large class of multi-component receptors that are activated by protein ligands known as cytokines and Growth Factors (GFs) - is at present only rudimentary. In previous work we have studied RET, a receptor tyrosine kinase that is important in sustaining the survival of a key population of sensory neurons in the peripheral nervous system, as a model system for the development and application of methods for the quantitative study of GF receptor activation and signaling. RET is activated by a family of four neuronal growth factors, GDNF, Neurturin, Artemin (ART) and persephin, in conjunction with one of four membrane-bound co-receptors known as GFR1-4. The activated receptor is a pentameric non-covalent complex comprising one molecule of growth factor bound to two molecules of RET plus two molecules of a GFR. In published work we have established the sequence of steps by which RET, in conjunction with ART and GFR3, form an activated receptor complex on live cells, and have determined the equilibrium constants for all steps, including the steps subsequent to initial ligand binding that occur exclusively on the cell membrane. We were thereby able to develop a quantitative mathematical model that relates the distribution of receptor complexes on the cell surface at a given concentration of ART to the affinities of particular steps, revealing for the first time how specific functional properties of the receptor such as its sensitivity and dynamic range relate to the molecular details of the activation mechanism. The objectives of the proposed work are as follows: 1. we will measure how RET phosphorylation responds to variations in the level of available RET present on the cell surface. In addition to being a stringent test of our proposed mechanism and extending our understanding of this process, these experiments also constitute a novel approach to establishing whether receptor activation occurs by ligand induced dimerization versus allosteric activation of preformed receptor dimers. 2. We will establish the quantitative relationships by which assembly of the activated RET receptor complex on the cell membrane is coupled to proximal and distal steps in cell signaling and to the functional cellular response of cell survival. Specifically, we will (i) measure the amplitude (absolute number of molecules activated), the evolution and decay kinetics, and the intrinsic lifetimes of activated molecular states, for key signaling events downstream of RET in the Ras/MAPK, p38MAPK, Akt, Plc/PKC and JNK signaling pathways;(ii) establish which signaling parameters at each step (instantaneous amplitude, peak amplitude, cumulative number of events over a given period, lifetime, etc.) are critical in driving the amplitude and sensitivity of the cell survival response to RET stimulation; (iii) establish the molecular mechanism responsible for the progressive signal sensitization that is observed from RET activation through to the cell survival response;and (iv) determine whether divergent signaling pathways are coupled similarly or differently to the level of activated RET present on the cell. 3. We will compare the mechanism by which ART and GFR3 bring about RET activation, established in our prior work, with the mechanism utilized by the alternative ligand/co-receptor pair GDNF/GFR1. We will additionally determine whether there are functionally significant differences in the signaling properties of the activated receptor complexes that result. If successful, the proposed work will result in a mechanistic and quantitative understanding of RET activation and signaling that is unprecedented for any other growth factor receptor, and will provide methods and approaches that can be applied to a wide range of other multi-component receptor systems. PUBLIC HEALTH RELEVANCE: The signals that cells send each other to regulate critical functions such as cell growth, maturation, and cell death are primarily mediated by a large and diverse family of messenger proteins known as cytokines and growth factors (GFs). These proteins are secreted into the intracellular medium, and are recognized by cells that possess appropriate 'receptor'proteins on their surface. The receptors become activated upon GF binding, leading to initiation of an intracellular signaling cascade that alters the behavior of the receiving cell in specific ways. Many important drugs, including Enbrel(R), Remicade(R), Humira(R), Herceptin(R), Avonex(R), Tarceva(R), and Iressa(R), treat a variety of cancers, autoimmune diseases and neurological disorders by disrupting GF receptor signaling. The hundreds of GFs and their receptors that have not so far been successfully targeted by approved drugs remain a fertile ground for new experimental therapies. In recent decades we have learned a great deal about the different proteins that function as cytokines or as part of cytokine receptors, and about the combinations in which they interact. However, in comparison with other medicinally important protein classes - such as enzymes, ion channels and G protein-coupled receptors - we still know remarkably little about how GF receptors perform their function. In particular, in almost no case do we understand the quantitative details of how GF binding is coupled to receptor activation, or receptor activation is in turn coupled to intracellular signaling, making it impossible to say, for example, how the properties of a given receptor lead a cell to respond to a particular level of GF in its environment. The proposed work aims to address this important gap in our knowledge by using the GF receptor RET, which is important in sustaining the survival of a key population of nerve cells in the spinal cord, as a model system to elucidate how GF/GF receptor interactions are coupled to intracellular signaling and to the resulting cellular response. This work builds directly upon prior successful work with the RET receptor in the PI's lab. If successful, the new knowledge and experimental methods it will deliver have the potential to contribute new and improved approaches to discovering and developing drugs that target GFs and their receptors.